Anti-human NGF antibody

ABSTRACT

[Task] To provide an anti-human NGF antibody or an antigen-binding fragment thereof that is excellent in safety by reducing the risk of side effects such as effects on a fetus and thrombus formation while maintaining high neutralizing activity, and to provide means for preventing or treating various diseases in which human NGF is involved in the formation of pathological conditions, by using the antibody or the antibody-binding fragment thereof. 
     [Means for Resolution] An anti-human NGF antibody Fab′ fragment comprising a heavy-chain variable region consisting of an amino acid sequence shown by SEQ ID NO:6 and a light-chain variable region consisting of an amino acid sequence shown by SEQ ID NO:4.

CROSS REFERENCE TO RELATED APPLICATION

This application is a National Stage entry under 35 USC 371 of PCT/JP2012/070433, filed on Aug. 10, 2012 , and herein incorporated in itsentirety by reference. Furthermore, this application claims priority toJapanese Patent Application No. 2011-176209 filed on Aug. 11, 2011 andJapanese Patent Application No. 2011-269215 filed on Dec. 8, 2011 , bothof which are herein incorporated in their entirety by reference.

TECHNICAL FIELD

The present invention relates to a novel anti-human NGF antibody. Morespecifically, the present invention relates to a Fab′ fragment of ananti-human NGF antibody.

BACKGROUND ART

A nerve growth factor (NGF) is one of humoral factors called generally“neurotrophic factors”, and plays an important role in generation anddifferentiation of neurons and in maintaining functions of neurons inthe body. As NGF receptors, a high affinity trkA receptor (receptor-typetyrosine kinase) and a low affinity p75NTR receptor are known. There isa report reporting that among these, the p75NTR binds to all of theneurotrophic factors and is involved in apoptosis in the process ofneuronal generation. However, the role of the p75NTR has not yet beensufficiently explained. Meanwhile, it is known that knockout mice of theNGF and the trkA receptor express the same phenotype (Non-PatentDocument 1), and it is considered that the physiological action of NGFis expressed mainly via the trkA receptor.

In 1993, there was a report reporting that the administration of NGF torats induced pain (Non-Patent Document 2), and since then, there hasbeen a report reporting that intravenous administration of NGF to humanbeings induces systemic myalgia and that topical administration of NGFexerts a systemic effect and induces hyperpathia and allodynia in aninjection site (Non-Patent Document 3). In addition, there is a reportreporting that a knockout mouse of the trkA receptor shows analgesia(Non-Patent Document 4), so it is considered that NGF is a moleculedeeply involved in the expression of pain. Regarding the correlationbetween NGF and the pathological condition of human pain, it has beendemonstrated that expression of NGF/trkA is accelerated in articularcartilages with osteoarthritis (OA) (Non-Patent Document 6) and that thelevel of NGF is increased in patients with rheumatoid arthritis(Non-Patent Document 7) or interstitial cystitis (Non-Patent Document8).

From the above facts, it is expected that if a monoclonal antibody whichspecifically binds to NGF and has an inhibitory activity against theaction of NGF can be developed, this will be useful for treating,preventing, and diagnosing various diseases including pain relating toNGF.

As anti-human NGF antibodies which have been clinically developed sofar, tanezumab (Patent Document 1) and PG110 (Patent Document 2) ashumanized anti-human NGF antibodies, and REGN475 (Patent Document 3),fulranumab (Patent Document 4), and MEDI-578 (Patent Document 5) asfully human anti-human NGF antibodies have been reported. Among these,tanezumab is being most briskly developed by priority, and there is areport reporting that according to clinical test results, this antibodyexerts a potent and extensive analgesic effect on pain such asarthralgia accompanied by osteoarthritis, chronic back pain, andcystalgia accompanied by interstitial cystitis (Non-Patent Documents 9to 11).

Generally, as main factors determining an effective dose of an antibodydrug, the neutralizing activity of an antibody against an antigen andthe amount of antigens present in the body are exemplified. Improvingthe neutralizing activity leads to the decrease of dose, andconsequently, this can be mentioned as very useful amelioration leadingto decrease in the financial burden of patients and medical costs. Ifthe decrease in dose can be realized, subcutaneous administration canalso be carried out. Subcutaneous administration has a major advantagethat a patient can perform self-injection at home if certain conditionsare satisfied. In addition, while the antibody drug is generallyadministered via drips for a certain time in many cases in theintravenous administration, the drug can be administered as a bolus inthe subcutaneous administration, which is another advantage. Both thephysician and the patient can select a preparation for intravenousadministration and a preparation for subcutaneous administration, andthis is a desirable factor. However, in the subcutaneous administration,a dose that can be given per administration is as small as about 1 mL ingeneral, so a sufficient amount of antibodies need to be included in thedose so as to express the drug efficacy. Moreover, unlike theintravenous administration, bioavailability needs to be considered forthe subcutaneous administration. That is, in order to realize apreparation for subcutaneous administration, it is required to preparean antibody which exhibits excellent solubility and expresses asufficient drug efficacy even at a small dose. Accordingly, if anantibody which has a higher neutralizing activity against NGF comparedto the antibodies in the related art is obtained, this will be usefulfor treating diseases relating to NGF and for improving convenience ofthe treatment.

As described above, though NGF is an important factor for growth ofneurons, performing sufficient examination in terms of safety isnecessary in developing medical drugs that inhibit the function of NGF.Particularly, as one of the respects which should be examined in termsof safety, the effects on a fetus are exemplified. So far, regarding thefunctional inhibition of NGF, there have been reports reporting that NGFmutation is the cause of congenital analgesia (Non-Patent Document 5),and that in an animal experiment, when a pregnant guinea pig is causedto produce an autoantibody to NGF so as to inhibit NGF in the body, thenewborn guinea pig shows symptoms of analgesia (Non-Patent Document 12).Moreover, in a test using NGF- or trkA-deficient mice, it has beendemonstrated that deficiency of NGF action inhibits the growth ofneurons of sensory nerves and sympathetic nerves in an embryo(Non-Patent Documents 4 and 13). From these results, it is understoodthat NGF is an essential factor of neurodevelopment in the early stageof development. The NGF-related diseases also include diseases thatwomen at a child-bearing age suffer from at a high rate, such asinterstitial cystitis (half or more of the patients are 44 years old oryounger, and 90% of patients are females (Non-Patent Document 14)),chronic back pain (an average age of 40 to 50, and over 50% of patientsare females (Non-Patent Documents 15 to 17)), and migraine (a peak ageof onset ranges from 15 to 40 years, and 80% of patients are female(Non-Patent Document 18)). In this situation, in developing the anti-NGFantibody as a medical drug, it is very important to avoid the risk ofside effects on a fetus in pregnant women.

As another risk factor in a case of developing the anti-NGF antibody asa medical drug, immunocomplex (IC) formation is exemplified. Theimmunocomplex which is a combination of an antigen and an antibody isgenerally treated in a reticuloendothelial system such as the spleen orthe liver. However, it has been reported that when a pathologicalcondition such as immune abnormality is caused or when the size of theformed IC is large, the IC loses solubility, which relates to theincrease of the risk of thrombus formation and to the onset of nephritiscaused by the glomerular accumulation of the IC. Though IgG is abivalent antibody, when an antigen is polyvalent, the IC may havevarious sizes due to lattice formation. The size of the IC depends onthe amount of an antibody and an antigen and the ratio therebetween,affinity of an antibody, and the like. For example, an anti-VEGFantibody bevacizumab (product name: Avastin) is an IgG1 antibody, andthere is a report reporting that this antibody forms an IC by binding toa dimer VEGF and induces thrombus formation. Specifically, when Avastinand VEGF are administered to a human FcγRIIα receptor Tg mouse,formation of a pulmonary artery thrombus is observed (Non-PatentDocument 19). In addition, there is a report reporting that an arterialthrombus is formed at a higher rate in patients with metastatic cancerwho receive chemotherapy with Avastin treatment, compared to a placebogroup receiving only chemotherapy (Non-Patent Document 20). Since NGFalso forms a dimer in the body to exert physiological activity, it isdesirable to further improve safety by avoiding the risk of IC formationin developing a medical drug of the anti-NGF antibody.

For the above reasons, for treating or preventing various NGF-relateddiseases, it is very important to obtain an anti-NGF antibody which isexcellent in safety by reducing the risk of side effects such as theeffects on a fetus and thrombus formation while maintaining a highneutralizing activity.

RELATED ART Patent Document

[Patent Document 1] WO2004/058184

[Patent Document 2] WO2005/061540

[Patent Document 3] WO2009/023540

[Patent Document 4] WO2005/019266

[Patent Document 5] WO2006/077441

Non-Patent Document

[Non-Patent Document 1] Conover J C, et al, Rev Neurosci. 1997, 8:13-27.

[Non-Patent Document 2] Lewin G R, et al, J. Neurosci. 1993, 13:2136-48.

[Non-Patent Document 3] Petty B G, et al, Ann Neurol. 1994, 36:244-6.

[Non-Patent Document 4] Smeyne R J, et al, Nature. 1994, 368:246-9.

[Non-Patent Document 5] Indo Y, et al, Nat. Genet. 1996, 13:485-8.

[Non-Patent Document 6] Iannone F, et al, Rheumatology 2002, 41:1413-8.

[Non-Patent Document 7] Aloe L, et al, Clin Exp Rheumatol. 1997,15:433-8.

[Non-Patent Document 8] Lowe E M, et al, Br J. Urol. 1997, 79:572-7.

[Non-Patent Document 9] Lane N E, et al, N Engl J. Med. 2010,363:1521-31.

[Non-Patent Document 10] Evans R J, et al, J. Urol. 2011, 185:1716-21.

[Non-Patent Document 11] Katz N, et al, Pain. 2011, in press

[Non-Patent Document 12] Johnson E M Jr, et al, Science. 1980,210:916-8.

[Non-Patent Document 13] Crowley C, et al, Cell. 1994, 76:1001-11.

[Non-Patent Document 14] Payne C K, et al, J. Urol. 2007, 177:2042-9.

[Non-Patent Document 15] Manchikanti L, et al, Pain Physician. 2010,13:E279-92.

[Non-Patent Document 16] Wilkens P, et al, JAMA. 2010, 304:45-52.

[Non-Patent Document 17] Buynak R, et al, Expert Opin Pharmacother.2010, 11:1787-804.

[Non-Patent Document 18] Sakai F, et al, Cephalalgia. 1997, 17:15-22.

[Non-Patent Document 19] Meyer T, et al, J Thromb Haemost. 2009,7:171-81.

[Non-Patent Document 20] Scappaticci F A, et al, J Natl Cancer Inst.2007, 99:1232-9.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide an anti-human NGFantibody or an antigen-binding fragment thereof that is excellent insafety by reducing the risk of side effects such as effects on a fetusand thrombus formation while maintaining high neutralizing activity.

Solution to Problem

The present invention includes the following invention as medically orindustrially useful substances and methods.

[1] An anti-human NGF antibody Fab′ fragment comprising:

a heavy-chain variable region consisting of an amino acid sequence shownby SEQ ID NO:6; and

a light-chain variable region consisting of an amino acid sequence shownby SEQ ID NO:4

[2] The Fab′ fragment according to [1], wherein a heavy-chain constantregion of the Fab′ fragment is a human Igγ1 constant region.

[3] The Fab′ fragment according to [1], wherein a light-chain constantregion of the Fab′ fragment is a human Igκ constant region.

[4] The Fab′ fragment according to [1], wherein the heavy-chain constantregion of the Fab′ fragment is the human Igγ1 constant region, and thelight-chain constant region of the Fab′ fragment is the human Igκconstant region.

[5] The Fab′ fragment according to [1], comprising:

a heavy-chain fragment consisting of an amino acid sequence shown by SEQID NO:10, SEQ ID NO:14, or SEQ ID NO:16; and

a light chain consisting of an amino acid sequence shown by SEQ IDNO:12.

[6] The Fab′ fragment according to any one of [1] to [5], wherein theFab′ fragment is conjugated to polyethylene glycol.

[7] A polynucleotide comprising a sequence that encodes the heavy-chainfragment of the Fab′ fragment according to any one of [1] to [6].

[8] A polynucleotide comprising a sequence that encodes the light chainof the Fab′ fragment according to any one of [1] to [6].

[9] An expression vector comprising the polynucleotide according to [7]and/or [8].

[10] A host cell transformed with the expression vector according to[9].

[11] The host cell according to [10], which is selected from a groupconsisting of the following (a) and (b),

(a) a host cell transformed with an expression vector comprising apolynucleotide comprising a sequence that encodes the heavy-chainfragment of the Fab′ fragment according to any one of [1] to [6] and apolynucleotide comprising a sequence that encodes the light chain of theFab′ fragment; and

(b) a host cell transformed with an expression vector comprising apolynucleotide comprising a sequence that encodes the heavy-chainfragment of the Fab′ fragment according to any one of [1] to [6] andwith an expression vector comprising a polynucleotide comprising asequence that encodes the light chain of the Fab′ fragment.

[12] A method of producing the anti-human NGF antibody Fab′ fragmentaccording to any one of [1] to [6], comprising expressing an anti-humanNGF antibody Fab′ fragment by culturing the host cell according to [10]or [11].

[13] An agent for treating pain, which comprises the Fab′ fragmentaccording to any one of [1] to [6].

[14] The agent for treating pain according to [13], wherein the pain isosteoarthritis pain.

[15] A method for preventing or treating pain, comprising administeringthe Fab′ fragment according to any one of [1] to [6].

[16] The method according to [15], wherein the pain is osteoarthritispain.

[17] The Fab′ fragment according to any one of [1] to [6] for use inpreventing or treating pain.

[18] The Fab′ fragment according to [17], wherein the pain isosteoarthritis pain.

Advantage Effects of the Invention

The anti-human NGF antibody Fab′ fragment of the present invention isuseful for preventing or treating various diseases in which human NGF isinvolved in the formation of pathological conditions. Due to its highneutralizing activity, the anti-human NGF antibody Fab′ fragment of thepresent invention brings about excellent improvement in clinicalapplications, such as decreases in dose, widening of administrationintervals, and improvement of the method of administration (for example,a subcutaneous injection). Moreover, in reducing the risk of sideeffects such as the effects on a fetus and thrombus formation, theanti-human NGF antibody Fab′ fragment of the present invention issignificantly excellent in terms of safety and greatly contributes tothe prevention or treatment of various diseases in which human NGF isinvolved in the formation of pathological conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows temporal change in the amount of an antibody retained inthe sole of a collagen-induced arthritis mouse model.

DESCRIPTION OF EMBODIMENTS

Hereinbelow, the present invention will be described in detail.

The present inventors repeated creative examination to prepare ananti-human NGF antibody or an antigen-binding fragment thereof. As aresult, they succeeded in preparing an anti-human NGF antibody Fab′fragment which is excellent in safety by reducing the risk of sideeffects such as the effects on a fetus and thrombus formation whilemaintaining high neutralizing activity.

The basic structure of an antibody molecule is common among therespective antibody classes and is constituted with a heavy chain havinga molecular weight of 50000 to 70000 and a light chain having amolecular weight of 20000 to 30000. The heavy chain generally consistsof a polypeptide chain including about 440 amino acids, and each classhas its characteristic structure. The heavy chains are called γ, μ, α,δ, and ε chains corresponding to IgG, IgM, IgA, IgD, and IgE.Furthermore, IgG has subclasses such as IgG1, IgG2, IgG3, and IgG4, andthese chains are called γ1, γ2, γ3, and γ4 respectively. A light chaingenerally consists of a polypeptide chain including about 220 aminoacids, and two types of the light chain including an L-type and a K-typelight chains are known, which are called λ and κ chains respectively.Regarding the peptide constitution of the basic structure of an antibodymolecule, two homologous heavy chains and two homologous light chainsare bound via disulfide bonds (S—S bonds) and non-covalent bonds, andthe molecular weight thereof is 150000 to 190000. The two kinds of lightchains can be paired with any heavy chain. Each antibody molecule alwaysconsists of two identical light chains and two identical heavy chains.

There are four intrachain S—S bonds in a heavy chain (five bonds for μand ε chains) and two in a light chain. One loop is formed per 100 to110 amino acid residues, and this steric structure is similar among therespective loops and is called a structural unit or a domain. For bothheavy chains and light chains, the amino acid sequence of the domainpositioned at the N-terminal thereof is not constant, even in areference standard from the same class (subclass) of the same animalspecies, and this domain is called the variable region. Each of thedomains is called a heavy-chain variable region (V_(H)) and alight-chain variable region (V_(L)) respectively. Since the amino acidsequence of the C-terminal side from the domain is almost constant ineach class or subclass, this region is called a constant region, andeach of the domains is described as C_(H)1, C_(H)2, C_(H)3 and C_(L),respectively.

The antigenic determinant site of an antibody is constituted with V_(H)and V_(L), and the binding specificity depends on the amino acidsequence of this site. On the other hand, biological activities such asbinding to complements or various cells reflect the differences in theconstant region structure among the various classes of Ig. It is knownthat the variability in the variable regions of the heavy chain andlight chain is mostly limited to three small hypervariable regionspresent in both chains, and these regions are called complementaritydetermining regions (CDRs; CDR1, CDR2 and CDR3 starting from theN-terminal side). The remaining portion of the variable region is calleda framework region (FR) and is relatively constant.

A region between the C_(H)1 domain and the C_(H)2 domain of theheavy-chain constant region of an antibody is called a hinge region.This region includes lots of proline residues and has a plurality ofinter-chain S—S bonds connecting two heavy-chains. For example, eachhinge region of human IgG1, IgG2, IgG3, and IgG4 includes 2, 4, 11, and2 cysteine residues respectively which constitute the inter-heavy-chainS—S bonds. The hinge region is a region highly sensitive to aproteolytic enzyme such as papain or pepsin. When an antibody isdigested with papain, its heavy chain is cleaved at a position closer tothe N-terminal side than to the inter-heavy-chain S—S bond of the hingeregion, whereby the antibody is broken down into two Fab fragments andone Fc fragment. The Fab fragment is constituted with a light-chain anda heavy-chain fragment including a heavy-chain variable region (V_(H)),a C_(H)1 domain, and a portion of the hinge region. When an antibody isdigested with pepsin, its heavy-chain is cleaved at a position closer tothe C-terminal side than to the inter-heavy-chain S—S bond of the hingeregion, whereby F(ab′)₂ fragments is generated. The F(ab′)₂ fragment isa fragment having a dimeric structure in which two Fab′ fragments bindto each other via the inter-heavy-chain S—S bond in the hinge region.The Fab′ fragment is constituted with a light-chain and a heavy-chainfragment including a heavy-chain variable region (V_(H)), a C_(H)1domain, and a portion of the hinge region. Cysteine residuesconstituting the inter-heavy-chain S—S bond are included in the portionof the hinge region. All of the Fab fragment, F(ab′)₂ fragment, and Fab′fragment include the variable region and have antigen-binding activity.

The anti-human NGF antibody Fab′ fragment of the present invention thatthe present inventors successfully prepared is a Fab′ fragment havingthe following characteristics.

The anti-human NGF antibody Fab′ fragment comprises a heavy-chainvariable region consisting of an amino acid sequence shown by SEQ IDNO:6 and a light-chain variable region consisting of an amino acidsequence shown by SEQ ID NO:4.

Specifically, the present inventors constructed antibodies using a humanmonoclonal antibody development technology, “VelocImmune” mouse[VelocImmune antibody technology; Regeneron Inc. (U.S. Pat. No.6,596,541)], and screened the antibodies using tests for variousbiological activities and physical properties, thereby succeeding inidentifying the anti-human NGF antibody Fab′ fragment of the presentinvention. In the VelocImmune technology, transgenic mice in which theendogenous immunoglobulin heavy and light chain variable regions arereplaced with the corresponding human variable regions are challengedwith the antigen of interest (for example, human βNGF), and lymphaticcells are recovered from the mice that express antibodies. The lymphaticcells are fused with mouse myeloma cells to prepare hybridomas. Thehybridoma cells are screened to identify hybridoma cells that producethose antibodies that specifically bind to the antigen of interest. Theantibodies that are produced herein are antibodies having the variableregions of human antibodies and the constant regions of mouse antibodies(also referred to as chimeric antibodies). Then, if the antibody thatbinds specifically to the antigen of interest and has a desiredneutralizing activity is identified, DNAs that encode the variableregions of the heavy chain and light chain of the antibody are isolatedfrom the hybridoma cells and linked to DNAs encoding the constantregions of the heavy chain and light chain of a desired class of humanantibody. The resulting gene encoding the heavy chain and light chain ofthe antibody is expressed in cells (e.g., CHO cells) to produce anantibody molecule. The heavy chain and light chain of the antibodyproduced by the above method are the heavy chain and light chain of a“fully human” antibody derived from a human immunoglobulin gene.

The anti-human NGF antibody Fab′ fragment of the present invention canbe easily prepared by those skilled in the art on the basis of thesequence information on the heavy-chain variable region and light-chainvariable region thereof disclosed herein, using a method commonly knownin the art. Preferably, the anti-human NGF antibody Fab′ fragment of thepresent invention can be prepared as a fully human antibody Fab′fragment by linking the heavy-chain variable region and light-chainvariable regions thereof to a part of the heavy-chain constant region(which includes C_(H)1 domain and a part of hinge region including hingeregion cysteine) and light-chain constant region of a human antibody,respectively. Specifically, a heavy-chain variable region gene fragmenthaving a base sequence that encodes the heavy-chain variable regionamino acid of the Fab′ fragment of the present invention (SEQ ID NO:6),and a light-chain variable region gene fragment having a base sequencethat encodes the light-chain variable region amino acid of the Fab′fragment of the present invention (SEQ ID NO:4) are prepared. Then, thevariable region genes of the heavy chain and light chain are linked toeach gene of a part of heavy-chain constant region and a light-chainconstant region in an appropriate class of human antibody to prepare afully human antibody Fab′ fragment gene. Next, this gene is linked to anappropriate expression vector and introduced into a cultured cell.Finally, this cultured cell is cultured, whereby a monoclonal Fab′fragment can be obtained from the culture supernatant.

The gene fragments that encode the heavy-chain and light-chain variableregion amino acids of the Fab′ fragment of the present invention can besynthesized using a gene synthesis method known in the art, on the basisof, for example, base sequences designed based on the amino acidsequences of the heavy-chain variable region and the light-chainvariable region. Examples of this gene synthesis method include variousmethods known to those skilled in the art, such as the antibody genesynthesis method described in WO90/07861.

Then, the above-described variable region gene fragments are linked tothe constant region gene of a human antibody to prepare a fully humanFab′ fragment gene. Although any subclass of the constant region (forexample, the constant region of a heavy chain such as the γ1, γ2, γ3 orγ4 chain, or the constant region of a light chain such as the λ or κchain) can be chosen as the constant region of the human antibody, humanIgγ1 as the heavy-chain constant region, and human Igκ as thelight-chain constant region, can preferably be used.

Subsequent to the preparation of this fully human antibody Fab′ fragmentgene, introduction of the gene into an expression vector, introductionof the expression vector into cultured cells, cultivation of thecultured cells, purification of the Fab′ fragment and the like can beperformed using various methods known in the art.

Examples of the expression vector that is linked to thus obtained geneinclude GS vector pEE6.4 or pEE12.4 (Lonza Biologics), but are notspecifically limited, so long as they can express such antibody gene.Also, an expression vector already having a human Ig constant regiongene such as AG-γ1 or AG-κ (for example, see WO94/20632) may be used.

The above-described expression vector is introduced into cultured cellsby, for example, a calcium phosphate method or an electroporation methodand the like.

Examples of the cultured cells into which the expression vector isintroduced include cultured cells such as CHO-K1SV cells, CHO-DG44 cellsand 293 cells, and these cells may be cultured by a conventional method.

The Fab′ fragment accumulated in a culture supernatant after culturingdescribed above can be purified by various types of columnchromatography. For example, it is possible to use column chromatographyusing KappaSelect or the like.

The Fab′ fragment of the present invention can be prepared using arecombinant expression method as described above. However, the Fab′fragment may be prepared by performing pepsin digestion after preparinga full-length antibody first, and treating the obtained F(ab′)₂ fragmentwith a reductant such as 2-mercaptoethanol.

Preferably, the anti-human NGF antibody Fab′ fragment of the presentinvention can be easily obtained by synthesizing DNA comprising a basesequence encoding the heavy-chain variable region amino acid sequenceshown by SEQ ID NO:6 and DNA comprising a base sequence encoding thelight-chain variable region amino acid sequence shown by SEQ ID NO:4,and linking the DNAs to a suitable class of human antibody constantregion genes, preferably a human Igγ1 constant region gene for the heavychain and a human Igκ constant region gene for the light chain, toconstruct a fully human antibody Fab′ fragment gene by using a methodknown in the art, and introducing the gene into an expression vector,introducing the expression vector into a cultured cell, culturing thecultured cell, and purifying an Fab′ fragment harvested from thecultured cell by using various methods known in the art. Preferably, DNAcomprising a base sequence encoding the heavy-chain variable regionamino acid sequences shown by SEQ ID NO:6 comprises the base sequencesshown by SEQ ID NO:5. Preferably, DNA comprising a base sequenceencoding the light-chain variable region amino acid sequences shown bySEQ ID NO:4 comprises the base sequences shown by SEQ ID NO:3.

In the present specification, the “Fab′ fragment” refers to a monovalentantibody fragment constituted with a light-chain and a heavy-chainfragment including a heavy-chain variable region (V_(H)), a C_(H)1domain, and a portion of a hinge region. In the portion of the hingeregion, at least one cysteine residue (also called a “hinge regioncysteine” in the present specification) other than cysteine residuesconstituting the S—S bond between the heavy chain and the light chain isincluded. The hinge region cysteine can be used as a modification siteof polyethylene glycol described later. The number of the hinge regioncysteines in the Fab′ fragment is variable within a range of from 1 toseveral cysteine residues depending on the class of an antibody used,and is easily adjustable by a person skilled in the art. For example,when a Fab′ fragment of a human IgG1 class (generally having two hingeregion cysteines in a hinge region) is prepared, a stop codon isinserted between a coding site of the first hinge region cysteine and acoding site of the second hinge region cysteine in the hinge region ofthe heavy chain, whereby a Fab′ fragment having one hinge regioncysteine in the hinge region can be prepared. In addition, if a stopcodon is inserted after the coding site of the second hinge regioncysteine, a Fab′ fragment having two hinge region cysteines in the hingeregion can be prepared.

The preferable heavy-chain fragment of the anti-human NGF antibody Fab′fragment of the present invention, comprising the heavy-chain variableregion consisting of the amino acid sequence shown by SEQ ID NO:6 and aportion of a human Igγ1 constant region, is a heavy-chain fragmentconsisting of the amino acid sequence shown by SEQ ID NO:10, SEQ IDNO:14, or SEQ ID NO:16. Preferably, DNA comprising a base sequence thatencodes the heavy-chain fragment of the anti-human NGF antibody Fab′fragment consisting of the amino acid sequence shown by SEQ ID NO:10,SEQ ID NO:14, or SEQ ID NO:16 comprises the base sequence shown by SEQID NO:9, SEQ ID NO:13, or SEQ ID NO:15. The preferable light chain ofthe anti-human NGF antibody Fab′ fragment of the present invention,comprising the light-chain variable region consisting of the amino acidsequence shown by SEQ ID NO:4 and a human Igκ constant region, is alight chain consisting of the amino acid sequence shown by SEQ ID NO:12.Preferably, DNA comprising a base sequence that encodes the light chainof the anti-human NGF antibody Fab′ fragment consisting of the aminoacid sequence shown by SEQ ID NO:12 comprises the base sequence shown bySEQ ID NO:11.

As a preferable anti-human NGF antibody Fab′ fragment of the presentinvention that comprises the heavy-chain fragment consisting of theamino acid sequence shown by SEQ ID NO:10 and the light-chain consistingof the amino acid sequence shown by SEQ ID NO:12, a fully human1-15(N52D) antibody Fab′ fragment described later in examples isexemplified. As a preferable anti-human NGF antibody Fab′ fragment ofthe present invention that comprises the heavy-chain fragment consistingof the amino acid sequence shown by SEQ ID NO:14 and the light chainconsisting of the amino acid sequence shown by SEQ ID NO:12, a fullyhuman 1-15(N52D-A) antibody Fab′ fragment described later in examples isexemplified. As a preferable anti-human NGF antibody Fab′ fragment ofthe present invention that comprises the heavy-chain fragment consistingof the amino acid sequence shown by SEQ ID NO:16 and the light chainconsisting of the amino acid sequence shown by SEQ ID NO:12, a fullyhuman 1-15(N52D-P) antibody Fab′ fragment described later in examples isexemplified.

The present invention also comprises an anti-human NGF antibody Fab′fragment that comprises the heavy-chain variable region comprising CDR1consisting of amino acid sequence at position from 31 to 35 of SEQ IDNO: 6, CDR2 consisting of amino acid sequence at position from 50 to 65of SEQ ID NO: 6, and CDR3 consisting of amino acid sequence at positionfrom 98 to 110 of SEQ ID NO: 6, and the light-chain variable regioncomprising CDR1 consisting of amino acid sequence at position from 24 to39 of SEQ ID NO: 4, CDR2 consisting of amino acid sequence at positionfrom 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of amino acidsequence at position from 94 to 102 of SEQ ID NO: 4. The Fab′ fragmentcan be also prepared by those skilled in the art according to proceduressuch as ones described above.

The anti-human NGF antibody Fab′ fragment of the present invention maybe modified by being conjugated to polyethylene glycol (PEG) via thehinge region cysteine thereof. PEG can be conjugated to the Fab′fragment by using methods known in the art (for example, EP0948544). Inthe present invention, linear or branched PEG having an arbitraryaverage molecular weight or a derivative thereof is usable, which can beeasily selected by a person skilled in the art according to the intendeduse. For example, in a tumor tissue or at the time of inflammatoryresponse, vascular permeability is markedly enhanced compared to anormal tissue, so substances reaching the tissue tend to leak out of theblood vessel and accumulate in the tumor or the inflammatory tissue (EPReffect). It is also known that a low molecular weight substance iseasily reabsorbed into blood vessels and that a high molecular weightsubstance is not easily reabsorbed. Therefore, in order to improveretentivity of the Fab′ fragment in a lesional tissue, PEG having a highaverage molecular weight (for example, about 40000 Da) may be conjugatedto this fragment. When the Fab′ fragment is desired to be rapidlyexcreted outside the body, PEG having a low average molecular weight(for example, about 10000 Da) may be conjugated to this fragment.Moreover, in order to facilitate the binding of PEG to the hinge regioncysteine, a PEG derivative may be used. For example, as described laterin examples, it is possible to use a PEG derivative to which athiol-reactive group such as maleimide has been bound and bind a thiolgroup of the hinge region cysteine to the maleimide group via a covalentbond. Generally, the average molecular weight of PEG ranges from about500 Da to about 50000 Da, preferably ranges from about 5000 Da to about40000 Da and more preferably ranges from about 10000 Da to about 40000Da.

The anti-human NGF antibody Fab′ fragment of the present invention bindsto human NGF. As the method of measuring binding activity of theobtained anti-human NGF antibody Fab′ fragment to the human NGF, thereis a method such as ELISA or FACS. For example, when ELISA is used,human βNGF is immobilized in an ELISA plate, the Fab′ fragment is addedthereto to cause a reaction, and then a secondary antibody such as ananti-kappa antibody labeled with an enzyme such as horseradishperoxidase (HRP) is allowed to react with the reaction mixture. Afterthe plate is washed, the activity is measured by using a reagent (forexample, a TMB chromogenic reagent in a case of HRP labeling) detectingthe activity, thereby identifying binding of the secondary antibody. Inaddition, the anti-human NGF antibody Fab′ fragment of the presentinvention also includes a Fab′ fragment that binds to NGF derived fromanother animal (for example, mouse NGF) as well as human NGF, so bindingactivity with respect to such a protein may be measured.

The anti-human NGF antibody Fab′ fragment of the present invention hasneutralizing activity with respect to human NGF. When being used in thepresent specification, the term “neutralizing activity” of theanti-human NGF antibody Fab′ fragment refers to an activity thatinhibits any biological activity resulting from NGF by binding to NGF,and the neutralizing activity can be evaluated using one or a pluralityof biological activities of NGF as an index. Examples of suchneutralizing activity include the inhibitory activity against binding ofNGF to trkA which is the NGF receptor, the inhibitory activity againstintracellular calcium influx mediated by an NGF-trkA signal, and theinhibitory activity against NGF-dependent cell survival signaling. Theneutralizing activity can be evaluated using methods described later inexamples.

In order to more specifically evaluate the effect of the anti-human NGFantibody Fab′ fragment of the present invention, an in vivo test may beperformed. For example, as described later in examples, the in vivo drugefficacy of the Fab′ fragment can be evaluated by an analgesic effecttest or the like that uses a mouse arthritis model. It is also possibleto evaluate the retention effect in a lesional tissue by using a testfor distribution property into a lesion.

Further, the anti-human NGF antibody Fab′ fragment of the presentinvention may also be evaluated in terms of the risk of side effects.For example, as described later in examples, by using a placentaltransfer test performed after administration of the Fab′ fragment topregnant animals, it is possible to evaluate the possibility that theanti-human NGF antibody Fab′ fragment of the present invention may exerteffects on a fetus. In addition, as described later in examples, thesize of an immunocomplex (IC) formed between the anti-human NGF antibodyFab′ fragment of the present invention and NGF is measured by using atest for IC formation with NGF, whereby the possibility of the inductionof thrombus formation can be evaluated.

In addition, as the method of evaluating various types of stability (forexample, thermal stability, long-term storage stability, andhigh-concentration stability) of the anti-human NGF antibody Fab′fragment of the present invention, a method of using differentialscanning calorimetry and a method of measuring the formation ofaggregates during storage are exemplified.

The anti-human NGF antibody Fab′ fragment of the present invention isoptionally purified and then formulated according to common methods, andcan be used for treating pain such as osteoarthritis pain (OA pain),rheumatic pain, cancer pain, neuropathic pain, chronic low back pain,postoperative pain, postherpetic neuralgia, painful diabetic neuropathy,fracture pain, and painful bladder syndrome and diseases in which NGF isinvolved in the formation of pathological conditions, such asinterstitial cystitis, acute pancreatitis, chronic pancreatitis, andendometriosis.

The anti-human NGF antibody Fab′ fragment of the present invention canbe used preferably as an agent for treating pain and more preferably asan agent for treating osteoarthritis pain. Examples of the formulationof this treating agent and the like include parenteral formulations suchas injectable agents and infusion agents, which are preferablyadministered by intravenous administration, subcutaneous administrationand the like. In the formulation process, carriers or additives thatmatch these formulations can be used within a pharmaceuticallyacceptable range.

The amount of inventive anti-human NGF antibody Fab′ fragment added inthe above-described formulation varies depending on the patient'ssymptom severity or age, the dosage form of the formulation used or thebinding titer of the antibody and the like; for example, about 0.001mg/kg to 100 mg/kg of the antibody may be used.

The present invention also provides a polynucleotide comprising asequence encoding the anti-human NGF antibody Fab′ fragment of thepresent invention, and an expression vector comprising the same. Thepresent invention also provides a polynucleotide comprising a sequenceencoding the heavy-chain variable region of the anti-human NGF antibodyFab′ fragment of the present invention, and a polynucleotide comprisinga sequence encoding the light-chain variable region of the anti-humanNGF antibody Fab′ fragment of the present invention, and expressionvector comprising either or both of them. The expression vector of thepresent invention is not specifically limited, so long as it can expressa gene that encodes the Fab′ fragment of the present invention or itsheavy-chain variable region and/or light-chain variable region invarious host cells of prokaryotic cells and/or eukaryotic cells, andproduce these polypeptides. Examples thereof include plasmid vectors,viral vectors (for example, adenovirus, retrovirus) and the like.Preferably, the expression vector of the present invention comprises apolynucleotide comprising either a sequence encoding the heavy chainfragment or light chain fragment of the above-described Fab′ fragment ofthe present invention, or both a polynucleotide comprising a sequenceencoding the heavy chain fragment of the Fab′ fragment of the presentinvention and a polynucleotide comprising a sequence encoding the lightchain of the Fab′ fragment of the present invention.

The expression vector of the present invention can comprise a gene thatencodes the anti-human NGF antibody Fab′ fragment of the presentinvention, or a gene that encodes the heavy-chain variable region and/orlight-chain variable region of the anti-human NGF antibody Fab′ fragmentof the present invention, and a promoter operably linked to the gene.Examples of a promoter for expressing a gene encoding the Fab′ fragmentof the present invention or its heavy-chain variable region and/orlight-chain variable region in a bacterium include Trp promoter, lacpromoter, recA promoter, λPL promoter, 1pp promoter, tac promoter andthe like, when the host is a bacterium of the genus Escherichia.Examples of a promoter for expression in yeast include PH05 promoter,PGK promoter, GAP promoter and ADH promoter, and some examples of apromoter for expression in the genus Bacillus include SL01 promoter,SP02 promoter, penP promoter and the like. When the host is a eukaryoticcell such as a mammalian cell, examples of the promoter includeSV40-derived promoter, retrovirus promoter, heat shock promoter and thelike.

When a bacterium, particularly Escherichia coli, is used as the hostcell, the expression vector of the present invention can furthercomprise an initiation codon, a stop codon, a terminator region and areplicable unit. When yeast, an animal cell or insect cell is used asthe host, the expression vector of the present invention can comprise aninitiation codon and a stop codon. In this case, it may comprise anenhancer sequence, noncoding regions on the 5′ side and 3′ side of agene that encodes the Fab′ fragment of the present invention or theheavy-chain variable region or light-chain variable region thereof, asecretion signal sequence, a splicing junction, a polyadenylationregion, a replicable unit or the like. Also, it may comprise a selectionmarker that is in common use (for example, tetracycline-resistant gene,ampicillin-resistant gene, kanamycin-resistant gene, neomycin-resistantgene, dihydrofolic acid reductase gene) according to the intended use.

The present invention also provides a transformant introduced with agene encoding the anti-human NGF antibody Fab′ fragment of the presentinvention or a gene encoding the heavy-chain variable region and/orlight-chain variable region of the anti-human NGF antibody Fab′ fragmentof the present invention. Such a transformant can be prepared by, forexample, transforming a host cell with the expression vector of thepresent invention. A host cell that is used to prepare the transformantis not specifically limited, so long as it is suitable for theaforementioned expression vector and is transformable; examples thereofinclude various cells such as natural cells or artificially establishedlines of cells commonly being used in the technical field of the presentinvention (for example, bacteria (bacteria of the genus Escherichia,bacteria of the genus Bacillus), yeasts (the genus Saccharomyces, thegenus Pichia and the like), animal cells or insect cells (for example,Sf9) and the like. The transformation can be performed by any knownmethod per se.

Preferably, the transformant of the present invention is either a hostcell transformed with an expression vector comprising a polynucleotidecomprising a sequence encoding the heavy-chain variable region of theFab′ fragment of the present invention and a polynucleotide comprising asequence encoding the light-chain variable region of the Fab′ fragment,or a host cell transformed with an expression vector comprising apolynucleotide comprising a sequence encoding the heavy-chain variableregion of the Fab′ fragment of the present invention and an expressionvector comprising a polynucleotide comprising a sequence encoding thelight-chain variable region of the Fab′ fragment. More preferably, thetransformant of the present invention is either a host cell transformedwith an expression vector comprising a polynucleotide comprising asequence encoding the heavy chain fragment of the above-described Fab′fragment of the present invention and a polynucleotide comprising asequence encoding the light chain of the Fab′ fragment, or a host celltransformed with an expression vector comprising a polynucleotidecomprising a sequence encoding the heavy chain fragment of theabove-mentioned Fab′ of the present invention and an expression vectorcomprising a polynucleotide comprising a sequence encoding the lightchain of the Fab′ fragment.

The present invention also provides a method for producing theanti-human NGF antibody Fab′ fragment of the present invention,comprising expressing in a host cell a gene encoding the anti-human NGFantibody Fab′ fragment of the present invention or a gene encoding theheavy-chain variable region and/or light-chain variable region of theanti-human NGF antibody Fab′ fragment of the present invention, that is,using such a transformant. Preferably, the host cell that is used in theabove method is a host cell transformed with the above-describedexpression vector of the present invention, and it may separately orsimultaneously comprise a polynucleotide comprising a sequence encodingthe heavy-chain variable region of the Fab′ fragment of the presentinvention and a polynucleotide comprising a sequence encoding thelight-chain variable region of the Fab′ fragment.

When producing the anti-human NGF antibody Fab′ fragment of the presentinvention, the transformant may be cultured in a nutrient medium. Thenutrient medium preferably contains a carbon source and an inorganicnitrogen source or organic nitrogen source, which are required for thegrowth of the transformant. Examples of the carbon source includeglucose, dextran, soluble starch, sucrose and the like; examples of theinorganic nitrogen source or organic nitrogen source include ammoniumsalts, nitrates, amino acids, corn steep liquor, peptone, casein, meatextract, soybean cake, potato extract and the like. If desired, othernutrients (for example, inorganic salts (for example, calcium chloride,sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics(for example, tetracycline, neomycin, ampicillin, kanamycin and thelike) and the like) may be contained.

Culture of the transformant is performed by a method known per se.Culture conditions, for example, temperature, pH of the medium, andculture time are suitably selected. For example, when the host is ananimal cell, an MEM medium containing about 5% to 20% fetal bovine serum(Science, Vol. 122, p. 501, 1952), DMEM medium (Virology, Vol. 8, p.396, 1959), RPMI1640 medium (J. Am. Med. Assoc., Vol. 199, p. 519,1967), 199 medium (Proc. Soc. Exp. Biol. Med., Vol. 73, p. 1, 1950) andthe like can be used as the medium. The pH of the medium is preferablyabout 6 to 8, culture is normally performed at about 30° C. to 40° C.for about 15 to 72 hours, and aeration or agitation may be performed asnecessary. When the host is an insect cell, for example, Grace's mediumcomprising fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol. 82, p.8404, 1985) and the like can be mentioned, and the pH thereof ispreferably about 5 to 8. Culturing is normally performed at about 20° C.to 40° C. for 15 to 100 hours, and aeration or agitation may beperformed as necessary. When the host is a bacterium, an actinomyces,yeast, or a filamentous fungus, for example, a liquid medium comprisingthe above-described nutrient sources is appropriate. A medium having apH of 5 to 8 is preferable. When the host is E. coli, preferred examplesof the medium include LB medium, M9 medium (Miller et al., Exp. Mol.Genet, Cold Spring Harbor Laboratory, p. 431, 1972) and the like. Inthis case, culture can be normally performed at 14° C. to 43° C. forabout 3 to 24 hours, while aeration or agitation is performed asnecessary. When the host is a bacterium of the genus Bacillus,cultivation can be normally performed at 30° C. to 40° C. for about 16to 96 hours, while aeration or agitation is performed as necessary. Whenthe host is yeast, examples of the medium include Burkholder's minimalmedium (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980),and the pH of the medium is desirably 5 to 8. Culturing is normallyperformed at about 20° C. to 35° C. for about 14 to 144 hours, andaeration or agitation may be performed as necessary.

The anti-human NGF antibody Fab′ fragment of the present invention canbe recovered, preferably isolated and purified, from a culturedtransformant as described above. Examples of the method of isolation andpurification include methods based on differences in solubility, such assalting-out and solvent precipitation; methods based on differences inmolecular weight, such as dialysis, ultrafiltration, gel filtration, andsodium dodecyl sulfate-polyacrylamide gel electrophoresis; methods basedon differences in electric charge, such as ion exchange chromatographyand hydroxyl apatite chromatography; methods based on specific affinity,such as affinity chromatography; methods based on differences inhydrophobicity, such as reverse phase high performance liquidchromatography; methods based on differences in isoelectric point, suchas isoelectric focusing; and the like.

Although the present invention has been generally described above,specific examples are provided herein only for a better understanding ofthe present invention. These examples are for illustrative purposes onlyand do not limit the scope of the present invention.

EXAMPLES

In steps using a commercially available kit or reagent, experiments wereperformed according to the attached protocols unless otherwisespecified.

Example 1 Immunization of VelocImmune Mouse

An anti-human NGF antibody was obtained by immunizing VelocImmune mice.In order to increase diversity of the obtained antibody, the presentinventors examined a plurality of immunization methods, routes ofadministration, adjuvants, immunization periods, and the like. By usinga human βNGF (R&D Systems, Inc.) as an immunogen, the present inventorsexamined a method of immunization in which the human βNGF is used forimmunization by mixing it with an adjuvant after dissolution, and amethod of immunization in which the human βNGF is used by mixing it withan adjuvant after thermally denaturing (treating at 80° C. for 10minutes in a 0.5% SDS solution). As the route of administration, footpadadministration and intraperitoneal administration were examined. As theadjuvant, TiterMax Gold (CytRx Corporation), complete Freund's Adjuvant(Sigma), incomplete Freund's Adjuvant (Sigma), and RIBI Adjuvant (CorixaCorporation) were examined. As an immunoactivator to be added, CpGoligonucleotide and Aluminum Phosphate Gel (BRENNTAG) were examined. Asthe immunization period, 3 to 14 weeks were examined. After the animalswas immunized several times, blood was collected from the caudal vein ofthe mice, and the titer was monitored. In this manner, VelocImmune miceproducing an antibody binding to the human NGF were selected.

The titer was measured using the following standard ELISA method. Thehuman βNGF was added to a Maxisorp 384 plate (Nunc) at 10 ng/well andimmobilized by being incubated overnight at 4° C. The next day, theplate was washed once with a wash solution (TBST: a tris buffer (TBS)containing 0.05% Tween-20), a blocking agent (TBST containing 20%Blocking One (Nacalai Tesque, Inc.)) was then added thereto, and theplate was left to stand at room temperature for an hour. After the platewas washed once with the TBST wash solution, the collected blood wasserially diluted and added to the plate. After an hour of incubation atroom temperature, the plate was washed three times with the TBST washsolution, and an HRP-goat anti-mouse Ig antibody (Zymed) which was2000-fold diluted with the TBST wash solution containing 5% Blocking Onewas added thereto. After an hour of incubation at room temperature, theplate was washed three times with the TBST wash solution. The plate wassupplemented with a TMB chromogenic reagent (SUMITOMO BAKELITE CO., LTD)and left to stand at room temperature for 10 minutes, a stop solution (2mol/L sulfuric acid) was then added thereto to stop the reaction, and anabsorbance at 450 nm was measured.

Example 2 Preparation of Anti-Human NGF Antibody-Producing Hybridoma

The mice selected by confirming the increase in antibody titer werefinally immunized (intravenous or intraperitoneal administration of anantigen). The spleen, lymph node, or the like of the immunized mice wasextracted according to a normal method so as to collect lymphocytes, andthe lymphocytes were fused with mouse myeloma cells SP2/0, therebypreparing a hybridoma. The hybridoma was subjected to limiting dilutionand monocloning, and the antibody was purified from the supernatant byusing a protein A or protein G column (GE Healthcare Japan).

Example 3 Evaluation of NGF-trkA Binding Inhibition

The human βNGF (R&D Systems, Inc.) was allowed to react with EZ-LINK5-(biotinamido)pentylamine (Pierce) at room temperature for 30 minutesin a dark place to perform biotin labeling, and the excess biotin wasremoved using a desalting column, thereby obtaining biotin-labeled humanβNGF. In the following Examples 6 and 7, the prepared biotin-labeledhuman βNGF was confirmed to have the same biological activity as that ofthe original human βNGF.

Inhibitory activity was measured by the following method. The human trkA(R&D Systems, Inc.) was added to a white Maxisorp 384 plate (Nunc) at 60ng/well and immobilized by being incubated overnight at 4° C. The nextday, the plate was washed once with the TBST wash solution, a blockingagent (TBST containing 20% Blocking One (Nacalai Tesque, Inc.)) was thenadded thereto, and the plate was left to stand at room temperature foran hour. Subsequently, a mixture obtained by mixing the biotin-labeledhuman βNGF (0.2 μg/ml) prepared as above with the antibody prepared inExample 2 was added to the trkA-immobilized plate having undergoneblocking. After an hour of incubation at room temperature, the plate waswashed three times with the TBST wash solution, and alkalinephosphatase-labeled streptavidin (Pierce) was added thereto. After anhour of incubation at room temperature, the plate was washed three timeswith the TBST wash solution and then supplemented with APU4 (BioFx)which is a reagent detecting chemiluminescence, and the amount ofchemiluminescence was measured by an EnVision counter (PerkinElmer Co.,Ltd.).

Example 4 Evaluation of Species Cross-Reactivity

When an antibody has cross-reactivity with respect to a mouse βNGF, itis possible to perform drug efficacy evaluation in a mouse pathologicalmodel by using the antibody. Consequently, a biotin-labeled mouse βNGFwas prepared in the method of Example 3 by using a mouse βNGF (R&DSystems, Inc.), whereby the cross-reactivity of the antibody to themouse βNGF was evaluated.

Example 5 Evaluation of Binding Specificity

The binding specificity of the antibody to NGF was evaluated by usingthe ELISA method described in Example 1. Specifically, NT-3 as a familymolecule showing the highest homology to NGF was used. Human NT-3(PeproTech) was added to the plate in the method of Example 1 at 20ng/well and immobilized in the plate, thereby allowing performance ofevaluation.

Example 6 Evaluation of NGF-trkA Signaling Inhibition

The inhibitory activity of the antibody against NGF-trkA signaling wasevaluated. NGF increases intracellular calcium (Ca²⁺) concentration viatrkA as the NGF receptor. Generally, the change in Ca²⁺ concentrationcan be evaluated in the presence of a calcium indicator by using anintracellular calcium (Ca²⁺) concentration measurement system (FLIPR;Molecular Devices, LLC.).

The inhibitory activity was measured by the following method. HEK293cells (WO2009/054468) caused to stably express the human trkA weredispensed in a 96-well poly-D-lysine-coated plate (Becton, Dickinson andCompany, Japan) at 2×10⁴ cells/well the day before experiment andcultured overnight. The next day, the culture medium was replaced with aDMEM culture medium (containing 3.6 mM sodium hydroxide (NaOH) and 2.5mM probenecid (Sigma)) containing a calcium indicator (Fluo4-AM;Dojindo) and left to stand at 37° C. for 30 minutes. Thereafter, thecells were washed twice with a wash solution (Hank's balanced saltsolution) (HBSS) (20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3.6 mM sodium hydroxide, 2.5 mM probenecid(Sigma), and 0.1% bovine serum albumin), and the culture medium wasreplaced with this wash solution at 150 μl/well. The cell plate was setin the FLIPR. By operating the FLIPR, a mixed solution of the antibodyobtained in Example 2 and NGF was added to the plate at 50 μl/well(final NGF concentration of 100 ng/ml), and the change in intracellularCa²⁺ concentration was measured. The difference between maximum andminimum values of the change in intracellular Ca²⁺ concentration wascalculated and stored as measurement data.

Example 7 Evaluation of NGF-Dependent Cell Survival Signaling Inhibition

When PC 12 cells naturally expressing the trkA and p75 receptors arecultured in a serum-free condition, NGF enables the cells to survive forseveral days. By the following method, the inhibitory activity of theantibody against the NGF-dependent cell survival signaling wasevaluated.

PC 12 cells were seeded in a 96-well collagen-coated plate (ASAHI TECHNOCO., LTD.) at 1×10⁴ cells/well and incubated overnight in an F12Kculture medium (Invitrogen) containing 2.5% fetal bovine serum and 15%inactivated horse serum (Invitrogen) at 37° C. under 5% CO₂. The nextday, the culture medium was replaced with only F12K in a serum-freecondition. After an hour, the antibody and the human βNGF (finalconcentration of 50 ng/ml) were added thereto, followed by culturing for72 hours. Subsequently, the culture solution was removed by anaspirator, and cell viability was measured using a reagent (CellTiterGlo; Promega Corporation) quantitating endogenous ATP of cells.

Example 8 Preparation of Fab Fragment

A digestive enzyme papain-bound gel was added to 1 mg/ml of the antibodyby using a Fab preparation kit (Pierce), followed by treatment at 37° C.for 3 hours. The treated reaction solution was added to a protein Gcolumn (GE Healthcare Japan), cleaved Fc and unreacted IgG were removedby being adsorbed onto the column, and the eluted fraction wascollected, thereby obtaining Fab fragments. The obtained Fab fragmentswere evaluated by the tests described in Examples 3, 6, and 7.

As a result of the evaluation of Examples 3 to 8, it was confirmed thatthe antibody named 1-15 (chimeric antibody) had high neutralizingactivity, species cross-reactivity, and binding specificity andmaintained high neutralizing activity even though this antibody is inthe form of a monovalent antibody fragment.

Example 9 Determining Antibody Gene Sequence

For the identified antibody 1-15, the present inventors cloned genesencoding the heavy chains and light chains of the antibody fromhybridomas. Specifically, a hybridoma clone was prepared in an amount of1×10⁵ or more and suspended in RLT buffer which is included in RNeasyMini Kit (QIAGEN), and then the cells were shredded with QIAshredder(QIAGEN). Subsequently, RNA was extracted according to the protocol, andby using the extracted RNA as a template, cDNA was synthesized using aDNA amplification kit (SMARTer RACE cDNA Amplification Kit; Clontech).PCR was carried out using the obtained cDNA, thereby elongating andamplifying the variable region of the heavy chains and light chains.Sequence analysis was performed directly on the PCR products by using asequencer (ABI PRISM 3100; Applied Biosystems). In addition, the PCRproducts were recombined with a PCR product subcloning vector such aspCR3.1-TOPO (Invitrogen), followed by gene sequence analysis, therebydetermining the sequence.

The determined base sequence of the heavy-chain variable region of theantibody 1-15 is shown by SEQ ID NO:1, and the amino acid sequencethereof is shown by SEQ ID NO:2. Moreover, the base sequence of thelight-chain variable region of the antibody is shown by SEQ ID NO:3, andthe amino acid sequence thereof is shown by SEQ ID NO:4. The CDR1, CDR2and CDR3 of the heavy-chain variable region of the antibody 1-15 is aregion of position from 31 to 35, 50 to 65, and 95 to 102 of theheavy-chain variable region based on Kabat numbering, respectively,which consists of the amino acid sequence at position from 31 to 35, 50to 65, and 98 to 110 of SEQ ID NO:2, respectively. The CDR1, CDR2 andCDR3 of the light-chain variable region of the antibody 1-15 is a regionof position from 24 to 34, 50 to 56, and 89 to 97 of the light-chainvariable region based on Kabat numbering, respectively, which consistsof the amino acid sequence at position from 24 to 39, 55 to 61, and 94to 102 of SEQ ID NO:4, respectively.

Example 10 Preparation of Mutant of Glycosylation Site of VariableRegion

The amino acid (SEQ ID NO:2) of the heavy-chain variable region of theantibody 1-15 described above includes an N-type glycosylation motifsequence as N-X-(T/S). Specifically, in the heavy-chain variable regionshown by SEQ ID NO:2, Asn (N52) at position 52 based on Kabat numberingcorresponds to the glycosylation site. It is known that thoughglycosylation of an antibody occurs during cell culturing if theantibody has glycosylation site, the glycosylation depends on theculturing conditions or the host expressing the antibody. In otherwords, even among the same antibody-producing cells established, thedegree of glycosylation is likely to vary with the culturing conditions(such as the culture medium and cell density), which leads to apossibility that it may be difficult to obtain antibody drugs havinguniform quality. Therefore, the present inventors prepared 1-15(N52D)which was obtained by introducing a mutation to N52 in the heavy-chainvariable region of the antibody 1-15.

The base sequence of the heavy-chain variable region of the prepared1-15(N52D) is shown by SEQ ID NO:5, and the amino acid sequence thereofis shown by SEQ ID NO:6. The CDR1, CDR2 and CDR3 of the heavy-chainvariable region of the antibody 1-15(N52D) is a region of position from31 to 35, 50 to 65, and 95 to 102 of the heavy-chain variable regionbased on Kabat numbering, respectively, which consists of the amino acidsequence at position from 31 to 35, 50 to 65, and 98 to 110 of SEQ IDNO:6, respectively.

Example 11 Preparation of Fully Human Antibody Fab′ Fragment

By using the heavy-chain variable regions of 1-15 and 1-15(N52D)described above and the light-chain variable region of 1-15, therespective fully human antibody Fab′ fragments were prepared.

A signal sequence was linked to the 5′ side of the respectiveheavy-chain variable region genes of 1-15 and 1-15(N52D), and theconstant region gene of human Igγ1 (Man Sung Co. et al., (1992) J.Immunol. Vol. 148(4):1149-1154) was linked to the 3′ side thereofrespectively. This heavy-chain fragment gene was inserted into GS vectorpEE6.4 (Lonza Biologics). At this time, in order to express the genes asa Fab′ fragment, a stop codon was inserted after the codon of Cys atposition 226 (corresponding to Cys at position 230 in the amino acidsequence of SEQ ID NO:8 and SEQ ID NO:10 described later) based on theEU index in the heavy-chain constant region gene. In addition, a signalsequence was linked to the 5′ side of the light-chain variable regiongene of 1-15, and the constant region gene of human κ chain (Man SungCo. et al., described above) was linked to the 3′ side thereofrespectively. This light-chain gene was inserted into GS vector pEE12.4(Lonza Biologics).

The Fab′ fragment was expressed in two manners including transientexpression and constant expression. In the transient expression,FreeStyle 293 cells (Invitrogen) cultured in FreeStyle 293 ExpressionMedium (Invitrogen) at about 1,000,000 cells/mL were transfected withthe above-described GS vectors of the heavy-chain fragment and thelight-chain by using 293fectin (Invitrogen), followed by culturing forseven days. In the constant expression, both the GS vectors describedabove were cleaved with restriction enzymes NotI and PvuI, followed byligation by using a DNA ligation kit (TAKARA BIO INC), therebyconstructing a GS vector into which genes of both the heavy-chainfragment and the light-chain were inserted. This expression vectorencodes the heavy-chain fragment, the light chain, and glutaminesynthetase and was expressed by being transfected to CHO-K1-SV cells.After the vectors were expressed in the respective manners, the culturesupernatant was purified by using KappaSelect (GE Healthcare Japan),thereby obtaining the respective Fab′ fragments.

The base sequence of the heavy-chain fragment of the prepared fullyhuman 1-15 antibody Fab′ fragment (also referred to as 1-15-Fab′) isshown by SEQ ID NO:7, and the amino acid sequence thereof is shown bySEQ ID NO:8 respectively.

The base sequence of the heavy-chain fragment of the prepared fullyhuman 1-15(N52D) antibody Fab′ fragment (also referred to as1-15(N52D)-Fab′) is shown by SEQ ID NO:9, and the amino acid sequencethereof is shown by SEQ ID NO:10 respectively.

The light chain of the respective Fab′ fragments are the same and thebase sequence thereof is shown by SEQ ID NO:11, and the amino sequencethereof is shown by SEQ ID NO:12 respectively.

Example 12 Evaluation of Neutralizing Activity and Expression Level ofFully Human Antibody Fab′ Fragment

The 1-15-Fab′ and the 1-15(N52D)-Fab′ obtained in Example 11 wereevaluated by the tests described in Examples 3 and 6. In the test ofExample 3, IC50 of the 1-15-Fab′ and the 1-15(N52D)-Fab′ was 0.17 μg/mland 0.18 μg/ml respectively. In the test of Example 6, IC50 of the1-15-Fab′ and the 1-15(N52D)-Fab′ was 0.021 μg/ml and 0.018 μg/mlrespectively. From these results, it was confirmed that the neutralizingactivity of the 1-15(N52D)-Fab′ was maintained to almost the same degreeas that of the unmodified 1-15-Fab′, and that the neutralizing activitywas not influenced even if a mutation was introduced.

In addition, the respective Fab′ fragments were expressed by theconstant expression, and the amount of antibody produced in the culturesupernatant of a stable expression cell pool was measured. As a result,the concentrations of the respective culture supernatants of the1-15-Fab′ and the 1-15(N52D)-Fab′ were 86 mg/L and 106 mg/Lrespectively, which showed that the 1-15(N52D)-Fab′ is an antibodyproduced in a higher amount than the unmodified 1-15-Fab′.

Example 13 Preparation of PEGylated Fab′ Fragment and Evaluation ofNeutralizing Activity

Next, the present inventors introduced PEG to the 1-15(N52D)-Fab′. Afterbeing purified by KappaSelect, the Fab′ fragment was subjected to areduction reaction by using TCEP hydrochloride(Tris(2-carboxyethyl)phosphine HCl), whereby the Fab′ fragment was madeinto a PEGylatable structure.

Specifically, TCEP was added to a Fab′ fragment solution of which theconcentration was adjusted to 1.2 mg/ml by a 20 mM of sodium phosphatebuffer (pH 6.8), such that the TCEP became 1 mM, followed by a reactionat 37° C. for 2 hours, and then the resultant was diluted with a 20 mMsodium acetate buffer (pH 5.0) to adjust pH. This solution was adsorbedonto a cation exchange resin (SP-5PW; TOSOH CORPORATION) and subjectedto NaCl gradient elution, and the main peak was collected. The obtainedFab′ fragment was diluted with a 20 mM sodium phosphate buffer (pH 6.8)so as to yield 1 mg/ml, the pH was adjusted to 6.8, and then thesolution was left to stand at 4° C. for a night or longer so as to benaturally oxidized. 40 kDa PEG (SUNBRIGHT GL2-400MA; NOF CORPORATION)was added to the solution to yield a final concentration of 0.1 mM, andthe solution was left to stand at room temperature for 2 hours and thenat 4° C. overnight. Having a maleimide group on the terminal thereof,this PEG rapidly reacts with Cys (C226 based on EU index; Cys atposition 230 of SEQ ID NO:10) of the carboxyl terminal of theheavy-chain fragment. The solution was diluted with a 20 mM sodiumacetate buffer (pH 4.5) to adjust pH and then adsorbed again onto acation exchange resin (SP-5PW; TOSOH CORPORATION), the resultant wassubjected to NaCl gradient elution, and the main peak was collected. Theresultant PEGylated Fab′ fragment was purified. This PEGylated1-15(N52D)-Fab′ is also called 1-15(N52D)-Fab′-PEG.

The neutralizing activity of the non-PEGylated and PEGylated1-15(N52D)-Fab′ s was evaluated by the method shown in Example 3. As aresult, while IC50 of the 1-15(N52D)-Fab′ was 0.15 μg/ml, IC50 of the1-15(N52D)-Fab′-PEG was 0.12 μg/ml (in terms of Fab′ fragmentconcentration), whereby it was confirmed that the neutralizing activityof the 1-15(N52D)-Fab′ was not influenced even if PEG was added.

In addition, by using the method of Example 6, the 1-15(N52D)-Fab′-PEGwas compared to the anti-human NGF antibody tanezumab of the prior art,in terms of the neutralizing activity with respect to human and mouseNGFs. As a result, while IC50 of the 1-15(N52D)-Fab′-PEG was 0.051 μg/mlfor the human NGF and 0.069 μg/ml for the mouse NGF, IC50 of thetanezumab was 0.17 μg/ml for the human NGF and 0.23 μg/ml for the mouseNGF. Therefore, it was confirmed that the neutralizing activity of the1-15(N52D)-Fab′-PEG was about 3.3 times stronger than the tanezumab,with respect to any of the human and mouse NGFs.

Example 14 Analgesic Effect Test Using Mouse Model of Adjuvant-InducedArthritis

The present inventors evaluated an analgesic effect of the above1-15(N52D)-Fab′-PEG on a mouse model of adjuvant-induced arthritis.

The 1-15(N52D)-Fab′-PEG was intravenously administered (0.03 mg/kg, 0.1mg/kg, and 0.3 mg/kg; the dose was 10 mL/kg) to mice, and 1 mg/mLFreund's complete adjuvant (Sigma) was administered in an amount of 25μL to the hindlimb footpad to induce pain. 24 hours after the paininduction, a rearing behavior for 20 minutes was measured. Specifically,by using SUPERMEX spontaneous activity monitoring system (MuromachiKikai Co., Ltd.), the number of times of spontaneous rearing behavior ofthe mice was automatically measured for 20 minutes by using an infraredbeam sensor (Matson et al., JPET 320:194-201, 2007). As a comparativecontrol, a prior art antibody tanezumab was used. As a result, while theintravenous administration of the tanezumab produced an analgesic effectof ED50=0.27 mg/kg, the 1-15(N52D)-Fab′-PEG produced an analgesic effectof ED50=0.11 mg/kg which showed an effectiveness greater by about 3times.

Example 15 Rat Placental Transfer Test

The 1-15(N52D)-Fab′-PEG or the tanezumab was intravenously administered(100 mg/kg, the dose was 10 mL/kg) to female rats in the 17th day ofpregnancy. Three days later, antibody concentration in the blood of themother and fetus was measured.

The antibody concentration was measured in the following manner. Thehuman βNGF (R&D Systems, Inc.) was added to a MULTI-ARRAY Plate(Standard) 96 plate (Meso Scale Discovery) at 25 ng/well and immobilizedby being left to stand at room temperature for an hour. The plate waswashed three times with the TBST wash solution, and a blocking agent (1%casein TBS; Thermo Fisher) was added thereto and left to stand at roomtemperature for an hour. Subsequently, a blood sample obtained bydiluting blood collected over time was added to the humanβNGF-immobilized plate having undergone blocking. After the mixture wasreacted at room temperature for 60 minutes under stirring, the plate waswashed three times with the TBST wash solution, and then abiotin-labeled anti-human Kappa antibody (Immuno-Biological LaboratoriesCo., Ltd.) was added thereto. After the mixture was reacted for 60minutes at room temperature under stirring, the plate was washed threetimes with the TBST wash solution, and SULFO-TAG-labeled streptavidin(Meso Scale Discovery) was added thereto. After the mixture was reactedfor 60 minutes at room temperature under stirring, the plate was washedthree times with the TBST wash solution, Read Buffer T (Meso ScaleDiscovery) was added thereto, and the amount of electrochemicalluminescence was measured with SECTOR Imager 6000 (Meso ScaleDiscovery).

This test was performed on three mother rats. Three days later, theantibody concentration of the 1-15(N52D)-Fab′-PEG and the tanezumab inthe blood of the mother rats was 12.1 μg/ml and 7.1 μg/ml on averagerespectively. Meanwhile, regarding the antibody concentration in theblood of 3 fetuses extracted from each mother rat (9 fetuses in total),while the concentration of 1-15(N52D)-Fab′-PEG in the blood was 0.01μg/ml (quantitation limit) or less in all fetuses, the concentration oftanezumab in the blood was 5.39 μg/ml on average. That is, while thetanezumab was transferred to the fetus at a rate of 75.9%, the1-15(N52D)-Fab′-PEG was transferred to the fetus at a rate of 0.08%(detection limit) or less. These results suggested that the1-15(N52D)-Fab′-PEG is a medical agent which is excellent in safety byavoiding the risk of side effects caused in a fetus due to NGFinhibition.

Example 16 Formation of Immunocomplex (IC)

Whether or not the 1-15(N52D)-Fab′-PEG formed an IC, or how large thesize of the formed IC was evaluated. Specifically, 1 mg/ml of the1-15(N52D)-Fab′-PEG was mixed with the human βNGF (R&D Systems, Inc.) ata molar ratio of 1:1, followed by incubation at room temperature for 3hours, thereby forming an IC. The particle size and distribution of theIC in this reaction solution were measured using Zetasizer Nano(Malvern) as an instrument measuring dynamic light scattering. For theanalysis, a Zetasizer v6.01 (Malvern) was used, and the particle sizewas indicated by a value (d. nm) analyzed in terms of Intensity (%).

The measured particle sizes are shown in the following Table 1. In thisexperiment, the particle size of only the NGF was 6.2 nm on average. Inthe case of only the tanezumab, a peak size was shown at 11.7 nm. Whenthe IC formed by incubating the tanezumab and the NGF was measured, thepeak size shifted to 91.3 nm. On the other hand, when an antibody notbinding to the NGF was used as a control antibody, the peak size wasstill 11.7 nm. In consideration of the shifting width, it was assumedthat each of the tanezumab and the NGF became a macromolecule as acombination of a plurality of molecules, whereby a large-sized IC wasformed. Contrary to this, when IC formation of the 1-15(N52D)-Fab′-PEGand the NGF was measured, the peak size shifted from 18.1 nm to 24.4 nm.In consideration of the shifting width, this result reflected onlyone-to-one binding and suggested that lattice formation did not occur inthe 1-15(N52D)-Fab′-PEG.

TABLE 1 Particle size Sample Peak (d · nm) Average (d · nm) Control IgGIgG 11.7 12.8 IgG + rhNGF 11.7 12.6 Tanezumab IgG 11.7 13.0 IgG + rhNGF91.3 99.2 1-15(N52D)-Fab′- IgG 18.1 19.8 PEG IgG + rhNGF 24.4 26.0

Example 17 Distribution Property into Lesional Tissue

An emulsion including collagen (bovine joint-derived type 2 collagen, 10mg/mL; Collagen technique workshop) and a complete Freund's adjuvant(0.5 mg/mL; DIFCO) at a ratio of 1:1 was subcutaneously administered tothe ankle joint of male DBA/1 mice, thereby preparing collagen-inducedarthritis models. Four weeks after the induction of arthritis, theemulsion was administered again to cause arthritis. The degree ofdevelopment (score and the size of swelling) of the arthritis inhindlimbs was observed to group the mice. Fluorescent labeling wasperformed on 1 mg/mL PBS solutions of the 1-15(N52D)-Fab′-PEG and thetanezumab by using SAIVI™ Rapid Antibody Labeling Kit, Alexa Fluor(registered trademark) 680 (Life Technologies Corporation). Eachsolution was administered to the caudal vein at 2 mg/kg (N=4). Thefluorescence accumulated in the swollen footpad was analyzed for 50hours from an hour after the administration by using an IVIS Spectrum(Caliper/Xenogen), and the fluorescence intensity was indicated asnumerical values.

FIG. 1 shows temporal change of the amount of antibody retained in thesole. The 1-15(N52D)-Fab′-PEG more clearly showed the retention effectin a lesional tissue compared to the tanezumab, and this effect lastedfor 48 hours. From this result, the 1-15(N52D)-Fab′-PEG is considered toefficiently exert an analgesic effect, and it is expected that thisantibody will be able to exert an analgesic effect equal to or strongerthan the strength of the drug efficacy with a low dose. It is alsoexpected that the 1-15(N52D)-Fab′-PEG can be a medical agent excellentin safety since this antibody is selectively accumulated in a lesionalsite.

Example 18 Preparation of Amino Acid Adduct of Fab′ Fragment

In order to improve the efficiency of PEG introduction into the1-15(N52D)-Fab′, the present inventors prepared Fab′ fragments that wereobtained by adding two alanines (A) or prolines (P) after the Cysresidue in the carboxyl terminal of the heavy-chain fragment andperformed expression and purification. The same method as in Example 11was used to prepare these Fab′ fragments. In this method, the codon ofthe two alanines or prolines were inserted after the codon of the Cysresidue of the carboxyl terminal of the heavy-chain fragment of the1-15(N52D)-Fab′, and a stop codon was inserted after this codon.

The base sequence of the heavy-chain fragment of alanine-added1-15(N52D)-Fab′ (a fully human 1-15(N52D-A) antibody Fab′ fragment; alsoreferred to as a 1-15(N52D-A)-Fab′) is shown by SEQ ID NO:13, and theamino acid sequence thereof is shown by SEQ ID NO:14 respectively. Thebase sequence of the heavy-chain fragment of proline-added1-15(N52D)-Fab′ (a fully human 1-15(N52D-P) antibody Fab′ fragment; alsoreferred to as a 1-15(N52D-P)-Fab′) is shown by SEQ ID NO:15, and theamino acid sequence thereof is shown by SEQ ID NO:16 respectively. Thelight chain of the respective Fab′ fragments is the same as the lightchain of the 1-15(N52D)-Fab′, and the base sequence and the amino acidsequence thereof are shown by SEQ ID NO:11 and SEQ ID NO:12respectively.

Example 19 Preparation of PEGylated 1-15(N52D-A)-Fab′ and Evaluation ofNeutralizing Activity and Pharmacological Evaluation

40 kDa PEG was conjugated to the 1-15(N52D-A)-Fab′ in the same manner asin Example 13, thereby obtaining PEGylated 1-15(N52D-A)-Fab′(hereinbelow, also referred to as 1-15(N52D-A)-Fab′-PEG).

The neutralizing activity of the 1-15(N52D-A)-Fab′-PEG was evaluated inthe method described in Example 3. As a result, while IC50 of the1-15(N52D)-Fab′-PEG was 0.081±0.034 μg/ml, IC50 of the1-15(N52D-A)-Fab′-PEG was 0.074±0.021 μg/ml. In addition, IC50 of thetanezumab at this time was 0.410±0.099 μg/ml.

Next, the neutralizing activity was compared using the method describedin Example 6. As a result, while IC50 of the 1-15(N52D)-Fab′-PEG was0.061±0.011 μg/ml for human NGF, IC50 of the 1-15(N52D-A)-Fab′-PEG was0.064±0.028 μg/ml.

Moreover, the analgesic effect in the adjuvant-induced arthritis modelwas evaluated using the method described in Example 14. As a result, the1-15(N52D-A)-Fab′-PEG showed the analgesic effect with respect to thearthritis model.

From the above results, it was confirmed that even if two alanines areadded after the Cys residue of the carboxyl terminal, the neutralizingactivity and the pharmacological activity are not influenced.

Example 20 Evaluation of Binding Affinity of 1-15(N52D-A)-Fab′-PEG

Thermodynamics in binding of 1-15(N52D-A)-Fab′-PEG and tanezumab to anNGF antigen was examined by Isothermal titration calorimetry (ITC)(Scappaticci F A, J Natl Cancer Inst. 2007, 99:1232-9. Velazquez-Compoy,A., et al, Curr Protoc Cell Biol. 2004, Chapter 17, Unit 17-18). Theentire measurement was performed using Auto-iTC 200 manufactured by GEhealthcare. During the experiment, a test was performed at the followingconcentration so as to evaluate binding of a monovalent Fab′ fragment toone molecule of antigen, and the entire test was performed in a PBSsolution. Specifically, 44 μM of human βNGF (R&D Systems, Inc.)contained in a titration syringe was titrated to calorimeter cellsfilled with an antibody sample (3 μM of 1-15(N52D-A)-Fab′-PEG or 1.5 μMof tanezumab) at 1.4 μL for 30 times, and the amount of heat producedthereby was detected. The obtained data was analyzed by a Single sitebinding model by using software attached to the instrument, wherebybinding affinity (Kd), a binding ratio (n), binding free energy (ΔG),binding enthalpy (ΔH), and binding entropy (-TΔS) associated withantigen-antibody binding were estimated. The results are shown in Table2.

As a result, while the value of Kd of tanezumab was 20.41 nM, the valueof Kd of 1-15(N52D-A)-Fab′-PEG was 1.49 nM, which showed that thebinding affinity of 1-15(N52D-A)-Fab′-PEG was stronger by 10 times ormore than that of tanezumab (Table 2).

TABLE 2 Kd (nM) ΔG (cal/mol) ΔH (cal/mol) −TΔS (cal/mol) Tanezumab 20.41−10490 −4759 −5731 1-15(N52D-A)- 1.49 −12041 −20806 8765 Fab′-PEG

Example 21 Preparation of PEGylated 1-15(N52D-A)-Fab′ Having Various PEGSizes and Evaluation of Neutralizing Activity

The 1-15(N52D-A)-Fab′ prepared in Example 18 was conjugated to 5 kDa PEGor 10 kDa PEG by using the similar procedure to that of Example 13.Specifically, Fab′ fragment solution prepared by using 20 mM Tris-HClbuffer (pH 7.4) was subjected to reduction treatment by using TCEP.Then, Fab′ fragment was collected by using a desalting column. PEG(SUNBRIGHT GL2-SOMA or SUNBRIGHT GL2-100MA; NOF CORPORATION) was addedto the obtained Fab′ fragment, and the solution was left to stand at 4°C. for a night. The 1-15(N52D-A)-Fab′ fragments conjugated to 5 kDa PEGor to 10 kDa PEG which were obtained in this manner are called1-15(N52D-A)-Fab′-SkPEG and 1-15(N52D-A)-Fab′-10 kPEG, respectively.

Thereafter, by using the method shown in Example 6, the respectivePEGylated Fab′ fragments were compared with each other in terms of theneutralizing activity. As a comparative control, the1-15(N52D-A)-Fab′-PEG (conjugated to 40 kDa PEG; hereinbelow, alsoreferred to as 1-15(N52D-A)-Fab′-40 kPEG) prepared in Example 19 wasused. At this time, the test was performed at a final NGF concentrationof 50 ng/ml. As a result, IC50 of the 1-15(N52D-A)-Fab′-5 kPEG,1-15(N52D-A)-Fab′-10 kPEG, and 1-15(N52D-A)-Fab′-40 kPEG were 0.030μg/ml, 0.028 μg/ml, and 0.023 μg/ml, respectively. From these results,it was understood that a PEG size ranging from 5 kDa to 40 kDa did notinfluence the neutralizing activity of Fab′ fragments.

Example 22 Mouse PK Evaluation for PEGylated 1-15(N52D-A)-Fab′ HavingVarious PEG Sizes

Mouse PK evaluation was performed for various types of PEGylated1-15(N52D-A)-Fab′. Specifically, 0.3 mg/kg of various types of PEGylated1-15(N52D-A)-Fab′ were intravenously administered, and blood wascollected 1, 4, 8, 12, 24, 48, 72, 96, and 168 hours after theadministration. The amount of tested antibody in the obtained blood wasmeasured by using the sandwich ELISA. Specifically, the tested antibodywas added to MSD plate (Meso Scale Discovery) which NGF was immobilized.The antibody bound to the plate was recognized by a biotin-labeledanti-human Kappa antibody, which was then detected by SULFO-TAG-labeledstreptavidin. The concentration of the antibody in the blood wascalculated by creating a calibration curve by using the respectivestandards. From the calculated concentration of the antibody in theblood, the antibody half-life in the blood (T1/2: hour) was calculated.As a result, T1/2 of the 1-15(N52D-A)-Fab′-5 kPEG, 1-15(N52D-A)-Fab′-10kPEG, and 1-15(N52D-A)-Fab′-40 kPEG were 13.8±2.2 hours, 17.7±0.4 hours,and 39.2±3.7 hours, respectively.

Example 23 Analgesic Effect Test Using Rat Plantar Incision Model

By using a rat post-plantar incision pain model (Brennan et al, CurrentProtocols in Pharmacology 2004; 5.34.1-5.34.8) which is considered toreflect postoperative pain in clinical practice, the analgesic effect ofthe 1-15(N52D-A)-Fab′-5 kPEG and the 1-15(N52D-A)-Fab′-10 kPEG onpostoperative pain was evaluated.

Specifically, 8 rats were assigned to each group, and the1-15(N52D-A)-Fab′-5 kPEG or -10 kPEG was intravenously administered tothe rats (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg, the dose was 1 mL/kg).Thereafter, in the sole of right hindlimb, a straight incision was madewhich extended 10 mm toward the toe from a starting point at a positiondistant by 5 mm from the end of the heel, and then mattress sutures wereimmediately made with a nylon thread at two sites, thereby inducingpain. Pain thresholds around the operation site were measured after 5hours and the first, second, third, fourth, and fifth days after thepain induction. For the measurement, a Dynamic plantar anesthesiometermanufactured by Ugo Basile was used to measure a pressure at which therats showed avoidance behavior when pressure was applied to the sole. Asa comparative control, the antibody tanezumab in the prior art was used.

As a result, while intravenous administration of tanezumab resulted inan analgesic effect of ED50=0.26 mg/kg on postoperative day 1, both the1-15(N52D-A)-Fab′-5 kPEG and -10 kPEG exerted an analgesic effect ofED50=0.15 mg/kg which was about twice as efficacious. In addition, asignificant analgesic effect of the 1-15(N52D-A)-Fab′-5 kPEG and -10kPEG was still observed on postoperative day 3 and 4, respectively.

Example 24 Evaluation of Aggregation Stability

The 1-15(N52D-A)-Fab′-40 kPEG was dissolved at 1 mg/ml and 10 mg/mlunder conditions of pH 5, pH 6, pH 7.4, and pH 9. Each of thesesolutions were placed under a condition of 50° C. so as to evaluateaggregation stability observed after 2 weeks. To evaluate theaggregation property, size exclusion chromatography was performed byusing Agilent 1100 manufactured by Agilent. As measurement conditions,0.1 M sodium phosphate containing 0.2 M arginine (pH 6.8) was used as abuffer of mobile phase, and TSK gel Super Sw3000 (TOSOH, 2.0 mm ID×300mm) was used as a column. The detection wavelength was 280 nm. In thetest at 1 mg/ml, tanezumab was used as comparative antibody, and theresults are shown in Table 3. In the test at 10 mg/ml, tanezumab andREGN 475 were used as comparative antibodies, and the results are shownin Table 4.

As a result, for tanezumab and REGN 475, marked increase in the amountof produced aggregates was observed after two weeks. Contrary to this,for the 1-15(N52D-A)-Fab′-40 kPEG, aggregates were almost not detected.This result suggests that the PEGylated 1-15(N52D-A)-Fab′ is highlylikely to be a drug having excellent storage stability.

TABLE 3 1-15(N52D-A)- Fab′-40kPEG Tanezumab Time Aggregation (%)Aggregation (%) pH (day) Polymer Dimer Polymer Dimer pH 5 0 0.0 0.7 0.22.5 14 0.0 0.7 11.7 5.6 pH 6 0 0.0 0.7 0.3 2.7 14 0.0 0.6 1.3 3.8 pH 7.40 0.0 0.7 0.3 2.8 14 0.0 0.6 3.1 3.8 pH 9 0 0.0 0.6 0.3 2.8 14 0.4 1.15.7 4.7

TABLE 4 1-15(N52D-A)- Fab′-40kPEG Tanezumab REGN 475 AggregationAggregation Aggregation Time (%) (%) (%) pH (day) Polymer Dimer PolymerDimer Polymer Dimer pH 5 0 0.0 0.4 0.9 4.8 0.3 1.7 14 0.1 0.5 23.5  8.48.7 3.9 pH 6 0 0.0 0.4 — — 1.2 3.0 14 0.0 0.7 — — 2.1 3.6 pH 7.4 0 0.00.5 1.7 6.1 0.3 2.0 14 0.0 0.8 5.6 7.8 4.0 2.6 pH 9 0 0.0 0.6 1.8 6.40.3 1.8 14 0.0 1.5 7.1 7.5 12.9 2.9 — not tested

INDUSTRIAL APPLICABILITY

The anti-human NGF antibody, more specifically, the anti-human NGFantibody Fab′ fragment of the present invention is useful for preventingor treating various diseases in which human NGF is involved in theformation of pathological conditions.

The invention claimed is:
 1. An anti-human NGF antibody Fab' fragmentcomprising: a heavy-chain fragment comprising a heavy-chain variableregion consisting of the amino acid sequence of SEQ ID NO:6; and a lightchain comprising a light-chain variable region consisting of the aminoacid sequence of SEQ ID NO:4.
 2. The Fab' fragment according to claim 1,wherein the heavy-chain fragment comprises a heavy-chain constant regionwhich is a human Igγ1 constant region.
 3. The Fab' fragment according toclaim 1, wherein the light chain comprises a light-chain constant regionwhich is a human Igκ constant region
 4. The Fab' fragment according toclaim 1, wherein the heavy-chain fragment comprises a heavy-chainconstant region which is a human Igγ1 constant region, and the lightchain comprises a light-chain constant region which is a human Igκconstant region.
 5. The Fab' fragment according to claim 1, comprising:a heavy-chain fragment consisting of the amino acid sequence of SEQ IDNO:10, SEQ ID NO:14, or SEQ ID NO:16; and a light chain consisting ofthe amino acid sequence of SEQ ID NO:12.
 6. The Fab' fragment accordingto claim 1, wherein the Fab' fragment is conjugated to polyethyleneglycol.
 7. The Fab' fragment according to claim 2, wherein the Fab'fragment is conjugated to polyethylene glycol.
 8. The Fab' fragmentaccording to claim 3, wherein the Fab' fragment is conjugated topolyethylene glycol.
 9. The Fab' fragment according to claim 4, whereinthe Fab' fragment is conjugated to polyethylene glycol.
 10. The Fab'fragment according to claim 5, wherein the Fab' fragment is conjugatedto polyethylene glycol.
 11. An expression vector comprising apolynucleotide comprising a sequence that encodes a heavy-chain fragmentcomprising a heavy-chain variable region consisting of the amino acidsequence of SEQ ID NO:6; and a polynucleotide comprising a sequence thatencodes a light chain comprising a light-chain variable regionconsisting of the amino acid sequence of SEQ ID NO:4.
 12. An isolatedhost cell transformed with the expression vector according to Claim 11.13. An isolated host cell, which is selected from the group consistingof: (a) a host cell transformed with an expression vector comprising apolynucleotide comprising a sequence that encodes a heavy-chain fragmentcomprising a heavy-chain variable region consisting of the amino acidsequence of SEQ ID NO:6 and a polynucleotide comprising sequence thatencodes a light chain comprising a light-chain variable regionconsisting of the amino acid sequence of SEQ ID NO:4; and (b) a hostcell transformed with an expression vector comprising a polynucleotidecomprising a sequence that encodes a heavy-chain fragment comprising aheavy-chain variable region consisting of the amino acid sequence of SEQID NO:6and with an expression vector comprising a polynucleotidecomprising a sequence that encodes a light chain comprising alight-chain variable region consisting of the amino acid sequence of SEQID NO:4.
 14. The isolated host cell according to claim 13, wherein saidhost cell is a host cell transformed with an expression vectorcomprising a polynucleotide comprising a sequence that encodes aheavy-chain fragment comprising a heavy-chain variable region consistingof the amino acid sequence of SEQ ID NO:6 and with an expression vectorcomprising a polynucleotide comprising a sequence that encodes a lightchain comprising a light-chain variable region consisting of the aminoacid sequence of SEQ ID NO:4.
 15. A method, comprising: expressing ananti-human NGF antibody Fab' fragment by culturing the host cellaccording to claim 13, thereby producing an anti-human NGF antibody Fab'fragment.
 16. A Fab' fragment produced by the method according to claim15.
 17. The Fab' fragment according to claim 16, wherein the Fab'fragment is conjugated to polyethylene glycol.